title: Rab1a and Rab5a preferentially bind to binary lipid compositions with higher stored curvature elastic energy.
abstract: Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.
authors.affiliation: Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: Marie L
authors.initials: ML
authors.lastname: Kirsten
authors.forename: Rudi A
authors.initials: RA
authors.lastname: Baron
authors.forename: Miguel C
authors.initials: MC
authors.lastname: Seabra
authors.forename: Oscar
authors.initials: O
authors.lastname: Ces
mesh_terms: D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008563:Membrane Lipids / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020761:rab1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008563:Membrane Lipids; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020761:rab1 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins